The time course and insulin concentration dependency of internalization of the insulin receptor have been examined in isolated rat adipose cells at 37 C. The internalization process was assessed by examining the subcellular distribution of [125I]insulin binding sites among plasma membrane, and high-density and low-density microsomal membrane fractions prepared by differential ultracentrifugation. A photoactive insulin analogue (B29-Napa-[125I]insulin), which specifically and covalenty labels the 138K insulin receptor subunit, has also been used to examine the effect of insulin on the subcellular distribution of insulin receptors. The photolabeled 138K receptor subunit in the plasma and low-density microsomal membrane fractions was quantitated by NaDodecylsulfate-polyacrylamide gel electrphoresis and autoradiography. A protocol was then developed for maintaining isolated rat adipose cells in primary tissue culture, and the acute insulin-induced internalization of its own receptor was examined by biosynthetically labeling cells with either [35S]methionine or [3H]glucosamine and immunoprecipitating the insulin receptors with anti-receptor antiserum. Finally, the internalization of the insulin receptor and spatial orientation of the 135K and 95K subunits of the receptor in the plasma membrane have been examined by labeling the receptor subunits through lactoperoxidase/Na125I iodination, a technique which side-specifically labels membrane proteins in intact cells and impermeable membrane vesicles, and immunoprecipitation. The results of these studies suggest that 1) insulin induces a rapid, and insulin concentration-and temperature-dependent translocation of both major subunits of its own receptor from the plasma membrane into at least two different intracellular membrane compartments associated respectively with the endoplasmic reticulum and Golgi-enriched membrane fractions, 2) this translocation occurs without receptor loss or alterations in receptor subunit structure, 3) the 135K receptor subunit is primarily, if not exclusively, exposed on the extracellular surface of the plasma membrane while the 95K receptor subunit traverses the membrane, and this vectorial disposition is inverted during translocation, and 4) this translocation represents internalization of the insulin receptor through and endocytic-like process.